1. Field of the Invention
The present invention relates to the field of molecular biology and nucleic acid chemistry. More specifically, it relates to methods and reagents for genotyping at the HLA Class I A locus. The invention therefore has applications in the fields of medicine generally, medical research and diagnostics specifically, transplantation biology, forensic science, and molecular biology.
2. Description of Related Art
The major histocompatibility complex (MHC) includes a number of genes that encode glycoproteins that, together with the T cell receptor (TCR), are the key elements of specificity in the T cell response to foreign and self antigens. There are two structurally distinct, but related, families of MHC molecules that present antigens to two subsets of T cells: Class 1 MHC molecules present antigens to T cells that express the CD8 cell surface glycoprotein, and Class II MHC molecules present antigens to T cells that express the CD4 cell surface glycoprotein. See Bjorkman and Parham, 1990, Ann. Rev. Biochem. 59:253-288, incorporated herein by reference. For a general review of the HLA Class II genes and proteins, see Trowsdale et al., 1985, Immunol. Rev. 85:5; and Giles and Capra, 1985, Adv. Immunol. 37:1, both incorporated herein by reference.
The Class I gene products function as restriction elements in the presentation of mainly endogenous pep tides to cytotexic T lymphocytes and are a major barrier for allogeneic tissue transplantation. Accurate determination of allelic subtypes is essential for typing potential transplantation donors, where very precise HLA matching of the donor and the transplant recipient appears to be critical in minimizing risk of rejection or graft versus host disease.
Significant advances have been made in developing DNA based typing methods for determining the HLA Class II genotype of an individual. The polymerase chain reaction is used to amplify a region nucleic acid encompassing a polymorphism and alleles are detected using sequence-specific oligonucleotide probes. For example, see Saiki et al., 1986, Nature 324:163; and Bugawan et al, 1988, J. Immunol. 141:4024-4030, both incorporated herein by reference. Some of these DNA based typing methods are now commercially available for forensic, research and clinical use.
In contrast to the advances made in the field of Class H HLA DNA typing, very little progress has been made in developing methods for Class I HLA DNA typing. One reason for this lack of progress is the complexity of the HLA Class I genes. The Class I genes encode the A, B, C, D, E, F, and G proteins, as well as other less well characterized products. The A, B, C, and E loci are known to encode approximately 41, 76, 18 and 4 different alleles, respectively. The F and G loci are currently not believed to be polymorphic. The currently known differences are primarily in the second and third exons of these genes (see Zemmour and Parham, 1991, Immunogenetics 33:310-320, incorporated herein by reference), although sequence variation in the fourth exon of these Class I genes is also known. See also Malissen et al., February, 1982, Proc. Natl. Acad. Sci USA 79:893-897, incorporated herein by reference.
Fernandez-Vina et al, 1992, Human Immunol. 33:163-173 describe a DNA typing system that uses sequence-specific amplification and detection by probe hybridization to distinguish 11 alleles of the HLA A2 and A28 groups. Browning et al., 1993, Proc. Natl. Acad. Sci USA 90:2842-2845 describe an HLA-A typing method which relies solely on sequence-specific amplification with a panel of primers. Oh et al, 1993, Tissue Antigens 41:135-142 describe an HLA-A typing system that includes amplification of part of the second and the third exon of the HLA-A locus (a region of HLA-H was co-amplified) and allele detection by hybridization with a panel of 28 probes. A number of isotypes previously defined by serology and isoelectric focusing could be defined by the patterns of hybridization. Each of these typing system is limited in the number of types that can be identified.
There is a need for a simple and rapid HLA-A typing system that can distinguish a greater number of alleles and genotypes.